Several patent documents and research articles are cited throughout this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these citations is incorporated by reference herein.
The most abundant transcripts in plastids are the ribosomal RNAs (rRNAs). The biosynthesis of plastid rRNA is highly regulated during development at both the transcriptional and posttranscriptional levels. In barley, rates of rrn transcription vary by 50-fold and rRNA stability by 35-fold in response to developmental and environmental cues (Baumgartner et al., 1993). Rates of rrn transcription were induced 10-fold in pea and tobacco chloroplasts in response to light (DuBell and Mullet, 1995; Shiina et al., 1998; Chun et al., 2001). Transcription of the plastid ribosomal RNA operon (rrn) in higher plants is from diverse promoters. The rrn operon in tobacco is transcribed by the multisubunit, plastid encoded RNA polymerase (PEP) from a sigma-70 type promoter (PrrnP1)(Vera and Sugiura, 1995) as in most higher plants including maize (Strittmatter et al., 1985), pea (Sun et al., 1989), carrot (Manna et al., 1994), rice (Silhavy and Maliga, 1998), barley (Hubschmann and Borner, 1998) and Arabidopsis (Sriraman et al., 1998a). In tobacco, in addition to the PrrnP1 PEP promoter, rrn is transcribed from a second promoter, PrrnP2, recognized by the nuclear-encoded plastid RNA polymerase (NEP) (Vera and Sugiura, 1995; Allison et al., 1996). In spinach, transcription of rrn initiates in the same region, but from a promoter distinct from the PrrnP1 or the PrrnP2 promoters. This promoter, Pc, is the only promoter upstream of the rrn operon in spinach and is probably also recognized by the NEP (Iratni et al., 1997; Bligny et al., 2000). Pc is utilized as a second rrn promoter in Arabidopsis (Sriraman et al., 1998a), and is recognized in mustard chloroplasts (Pfannschmidt and Link, 1997).
To identify promoter elements important for PrrnP1 function, promoter dissection was carried out in vivo and in vitro. In vivo dissection was carried out by studying expression of uidA reporter genes from an ordered set of PrrnP1 promoter derivatives (Staub and Maliga, 1993; Allison and Maliga, 1995). In vitro dissection was carried out by measuring transcript accumulation from mini-genes which consist of a PrrnP1 promoter derivative and transcription terminators (Jolly and Bogorad, 1980; Link, 1984; Gruissem and Zurawski, 1985; Orozco et al., 1985). In vivo dissection of the plastid rrn operon promoter indicates that sequences upstream of the conserved −35 box are important for promoter function.